DBR1 siRNA inhibition of HIV-1 replication

Abstract

Background: HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1) expression would specifically inhibit HIV-1 replication.

Results: We designed three short interfering RNA (siRNA) molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression.

Conclusion: These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.

Figure 1

Suppression of DBR1 mRNA expression by DBR1 siRNA. (A) GHOST-R5X4 cells were transfected with various concentrations of DBR1 siRNA pHyper-D123 (0.8 μg/ml, 1.6 μg/ml, 3.2 μg/ml, 6.4 μg/ml, 12.8 μg/ml) or mock transfected. Forty-eight hours post transfection, RNA was isolated, treated with Dnase I and used as template in real-time quantitative RT-PCR. Each reaction included 300 ng RNA, 0.5 μM each gene-specific primer for DBR1 and GAPDH amplification. GAPDH was used as an internal control. (B) GHOST-R5X4 cells were transfected with 12.8 μg/ml DBR1 siRNA pHyper-D1, pHyper-D2, pHyper-D3, pHyper-D123 or control pHyper and cells were collected at the indicated time points. RNA was isolated, treated with Dnase I and analyzed by real-time quantitative RT-PCR. Results shown are from triplicate samples in a representative experiment. Error bars indicate standard deviations. Results that are significant by Student's t-test are indicated by asterisks (p < 0.05).

Figure 2

Determination of cell viability. (A) GHOST-R5X4 cells were transfected with 3.2 μg/ml, 6.4 μg/ml, 12.8 μg/ml, 25.6 μg/ml or 51.2 μg/ml DBR1 siRNA pHyper-D123 (black bars) or the same concentrations of pHyper alone (white bars) as a control. (B) GHOST-R5X4 cells were transfected with 1 μg/ml, 4 μg/ml, 6 μg/ml CDM9-DBR1 (black bars) or the same concentrations of CDM9 alone (white bars) as a control. After forty-eight hours cellular viability was assayed with a commercial MTS assay kit (Promega). Results shown are from triplicate samples in a representative experiment. Error bars indicate standard deviations.

Figure 3

DBR1 siRNA inhibition of HIV-1 replication. GHOST-R5X4 cells were transfected with 12.8 μg/ml DBR1 siRNA pHyper-D1, pHyper-D2, pHyper-D3, pHyper-D123 or control pHyper. Forty-eight hours later, cells were infected with HIV-1 (JR-CSF) for twenty-four hours and p24 in supernatants was analyzed using a commercial HIV-1 p24 ELISA kit. All ELISA measurements were done in triplicate. Error bars indicate +/- standard deviation. Results marked with an asterisk are significant by Student's t-test (p < 0.05).

Figure 4

DBR1 over-expression does not affect HIV-1 replication. (A) GHOST-R5X4 cells were transfected with various amounts of CDM9-DBR1 (1 μg/ml, 4 μg/ml, 6 μg/ml) or mock transfected for forty-eight hours. Total RNA was isolated and DBR1 expression was quantified by real-time RT-PCR. (B) GHOST-R5X4 cells were transfected with CDM9-DBR1 (black bar, CDM9-DBR1), control vector CDM9 (white bar, CDM9-DBR1) or DBR1 siRNA pHyper-D123 (black bar, DBR1 siRNA), control vector pHyper (white bar, DBR1 siRNA), or cotransfected CDM9-DBR1 and DBR1 siRNA pHyper-D123 (black bar, CDM9-DBR1 + DBR1 siRNA), control vector CDM9 and pHyper (white bar, CDM9-DBR1 + DBR1 siRNA) for forty-eight hours and then infected with HIV-1 (JR-CSF) for twenty-four hours. Supernatants were collected and p24 was assayed using a commercially available HIV-1 p24 ELISA kit. All ELISA measurements were done in triplicate. Error bars indicate standard deviations. The asterisk denotes p < 0.05 by Student's t-test.

Figure 5

DBR1 siRNA suppresses HIV-1 reverse transcription after minus-strand strong-stop DNA formation. (A) Location of primers. The oligonucleotide primers M667/AA55 specific for the R/U5 region of the LTR were used to detect early reverse transcripts (strong-stop DNA) (B) and env primers Env1/Env2 to were used detect intermediate reverse transcripts (C). LTR/gag primers M667/M661 were chosen to detect complete first-strand viral cDNA (D). In B, C and D, GHOST-R5X4 cells were transfected with DBR1 siRNA pHyper-D123 (black bars, siRNA), control vector pHyper (white bars, siRNA) or CDM9-DBR1 (black bars, cDNA), control vector CDM9 (white bars, cDNA) or co-transfected with CDM9-DBR1 and DBR1 siRNA pHyper-D123 (black bars, siRNA + cDNA), control vectors CDM9 and pHyper (white bars, siRNA + cDNA) for forty-eight hours, followed by infection with Dnase-treated HIV-1 (JR-CSF). Twenty-four hours later, the cells were lysed and DNA was isolated for real time quantitative PCR to evaluate the synthesis of HIV-1 cDNA. Copies of HIV-1 DNA were normalized against copies of β-globin. Error bars indicate standard deviations. Asterisks denote p < 0.05 by Student's t-test.