Pyrosequencing of small non-coding RNAs in HIV-1 infected cells: evidence for the processing of a viral-cellular double-stranded RNA hybrid

Abstract

Small non-coding RNAs of 18-25 nt in length can regulate gene expression through the RNA interference (RNAi) pathway. To characterize small RNAs in HIV-1-infected cells, we performed linker-ligated cloning followed by high-throughput pyrosequencing. Here, we report the composition of small RNAs in HIV-1 productively infected MT4 T-cells. We identified several HIV-1 small RNA clones and a highly abundant small 18-nt RNA that is antisense to the HIV-1 primer-binding site (PBS). This 18-nt RNA apparently originated from the dsRNA hybrid formed by the HIV-1 PBS and the 3' end of the human cellular tRNAlys3. It was found to associate with the Ago2 protein, suggesting its possible function in the cellular RNAi machinery for targeting HIV-1.

Figure 1.

Pyrosequencing of small RNAs from HIV-1 infected T cells. (A) A pie chart indicates the distribution of different cellular small RNAs. (B) Distribution frequency showing the number of times that different miRNAs were cloned.

Figure 2.

Characterization of HIV-1 small non-coding RNAs. (A) TARncRNA (highlighted in yellow) maps to the LTR (Left). The predicted TAR hairpin structure and the location of the TARncRNA are shown. NEFncRNA, NEFncRNA* (highlighted in yellow and grey) and hiv1-miR-N367 (red) (9,35) are indicated in the Nef coding sequence (top). Two alternate hairpin structures can be predicted for these sequences. In one structure, NEFncRNA is complementary to the previously cloned NEFmiRNA, hiv-1-miR367. In the second structure, NEFncRNA is in a hairpin with NEFncRNA* sequence which was also cloned in our small RNA library. Both structures share similar calculated free energies. (B) Mfold (68) prediction of the HIV-1 pNL4-3 RRE structure (top). RRE stem I is the apparent precursor for a vsiRNA*-vsiRNA (red) (36) duplex and for the RREncRNA (highlighted in yellow). Both vsiRNA* and RREncRNA were cloned in the current study. Free energies (E) of the vsiRNA*-vsiRNA and RREncRNA structures are −38.0 and −34.2 kcal/mol, respectively.

Figure 3.

Quantification of HIV-1 small RNAs by real-time RT–PCR. The HIV-1 small RNAs (TARncRNA, RREncRNA and NEFncRNA) were verified by quantitative RT–PCR using U6 small RNA for normalization. Methodology in Supplementary Figure S1.

Figure 4.

Detection of PBSncRNA that is antisense to the HIV-1 PBS. (A) Multiple copies of PBSncRNAs were cloned from HIV-1 infected MT4 cells. The numbering corresponds to pNL4-3 sequence. (B) A schematic diagram showing the duplex formed between the HIV-1 PBS with the 3′-end of tRNAlys3. PBSncRNA sequence is highlighted in grey. (C) Immunoprecipitation of the AGO2 protein followed by primer-specific RT–qPCR of the AGO2-associated RNA shows that the amount of PBSncRNA quantified in 293T-AGO2 cells is dose-dependent on the amount of transfected pNL4-3 (pNL). (D) Northern blot analysis demonstrating increased detection of PBSncRNA in PMA-induced HIV-1 latently infected U1 cells, and in HIV-1 molecular clone (pNL4-3)-transfected HeLa cells (upper panel). Production of HIV-1 from the indicated cells was verified by measuring RT activity (bottom panel). (E) Quantification of the predicted processing of the 3′-end of the indicated tRNAs in HIV-1 infected MT4 cells. The relative detection of different 3′-end—tRNA fragments from the sequencing data is compared. The value of the 18-nt PBSncRNA from Lys(UUU) (tRNAlys3) was set as 1. tRNA(UUU) = tRNAlys3.

Figure 5.

Evidence for the in vitro processing and intracellular RISC-incorporation of PBSncRNA. (A) In vitro Dicer assay of the HIV-1 PBS–tRNA hybrid (the PBS containing radiolabeled transcript was made as described in Supplementary Figure S2) produced a small RNA of 18 nt (asterisk; lane 1). This small RNA was not observed in the absence of either tRNA (lane 2) or tRNA+Dicer (lane 3). As a positive control, in vitro T7 RNA polymerase synthesized and then annealed ds GFP RNA (T7dsGFP) was processed in vitro by Dicer, which yielded the expected small RNAs of ∼22 bp (lane 4). (B) Non-nucleoside RT inhibitor (Nevirapine) does not affect the expression of PBSncRNA in a single-cycle transfection of an HIV-1 molecular clone. We transfected pNL4-3 into nevirapine treated (+; lanes 5–8) or untreated cells (−; lanes 1–4) and subsequently quantified PBSncRNA. Similar expression levels of PBSncRNA was detected in nevirapine treated (lane 7) and untreated (lane 3) 293T-AGO2 cells. Controls include the immunopreciptation using anti-FLAG in control cell line (293T-cont) (lanes 1 and 5) and the attempted detection of a randomly selected HIV-1 sequence (HIVs3263) (lanes 2, 4, 6 and 8). (C) siRNA knock down of Dicer reduced the expression of PBSncRNA. PBSncRNA was quantified in 293T-AGO2 cells transfected with control-siRNA (si-cont) or Dicer-siRNA (si-Dicer) and co-transfected with pNL4-3. The amount of AGO2-associated PBSncRNA was then quantified by RT–qPCR after immunoprecipitation with anti-AGO2 (lanes 1 and 2). Control immunoprecipitation was also performed with an irrelevant IgG (lanes 3 and 4). (D) Immunoprecipitation (IP) of FLAG-Ago2 using anti-FLAG or a control IgG antibody was performed in a 293T-AGO2 cell line or in a control 293T parental cell line as described in the text. Quantitative real-time RT–qPCR detection of the HIV-1 PBSncRNA or two randomly chosen HIV-1 sequences (HIVs3263 and HIVs5543) from the IP products was performed. The value of the RT–qPCR result from the IP of 293T-AGO2 transfected with pNL4-3 was set as 1.

Figure 6.

Over expressed PBSncRNA or knock down of physiologically expressed PBSncRNA can modulate viral replication. (A) A schematic diagram of the LucPBS construct. A single copy of HIV-1 PBS sequence (TGGCGCCCGAACAGGGAC) was positioned downstream of the luciferase coding sequence. (B) Functional studies employing synthetic dsRNA oligonucleotide corresponding to the PBSncRNA or its mutated form, PBSncRNAmutant. Mutated nucleotides are boxed. Different concentrations (5 or 50 nM) of the PBSncRNA and PBSncRNAmutant were separately co-transfected with LucPBS construct (described in A) into HeLa cells. The PBSncRNA inhibited up to ∼80% the luciferase expression. (C) Similar experiments were performed in 293T cells using HIV-1 molecular clone (pNL4-3) in a single round replication assay (see text). The PBSncRNA effectively inhibited up to ∼80% of HIV-1 replication while no inhibition was observed with PBSncRNAmutant. (D) De-repression of HIV-1 replication by knock down of cell endogenous PBSncRNA using RNA ‘antagomirs’. Co-transfection of pNL4-3 and increasing amount of antagomirs, complementary to the PBSncRNA, in 293T cells increased HIV-1 replication as measured by RT activities.