Replication competent HIV-1 viruses that express intragenomic microRNA reveal discrete RNA-interference mechanisms that affect viral replication
© Klase et al; licensee BioMed Central Ltd. 2011
Received: 9 November 2011
Accepted: 23 November 2011
Published: 23 November 2011
It remains unclear whether retroviruses can encode and express an intragenomic microRNA (miRNA). Some have suggested that processing by the Drosha and Dicer enzymes might preclude the viability of a replicating retroviral RNA genome that contains a cis-embedded miRNA. To date, while many studies have shown that lentiviral vectors containing miRNAs can transduce mammalian cells and express the inserted miRNA efficiently, no study has examined the impact on the replication of a lentivirus such as HIV-1 after the deliberate intragenomic insertion of a bona fide miRNA.
We have constructed several HIV-1 molecular clones, each containing a discrete cellular miRNA positioned in Nef. These retroviral genomes express the inserted miRNA and are generally replication competent in T-cells. The inserted intragenomic miRNA was observed to elicit two different consequences for HIV-1 replication. First, the expression of miRNAs with predicted target sequences in the HIV-1 genome was found to reduce viral replication. Second, in one case, where an inserted miRNA was unusually well-processed by Drosha, this processing event inhibited viral replication.
This is the first study to examine in detail the replication competence of HIV-1 genomes that express cis-embedded miRNAs. The results indicate that a replication competent retroviral genome is not precluded from encoding and expressing a viral miRNA.
RNA interference (RNAi) is a regulatory mechanism conserved in organisms from protozoans to mammals [1–3]. This process employs a small single stranded RNA of 20-24 nucleotides in length which is used as a guide-RNA to direct an RNA-induced silencing complex (RISC) containing the argonaut protein and co-factors to the targeted RNA [4–8]. Human cells encode 1,527 miRNA genes  that are transcribed into precursor primary miRNAs (pri-miRNAs) which are processed in the nucleus by Drosha into shorter hairpin products called pre-miRNA. The pre-miRNAs are exported into the cytoplasm by Exportin-5, and cleaved by Dicer to generate 20-24 nucleotide RNA duplexes, one strand of which is loaded into the Argonaute containing RISC [10–14]. miRNA-RISC complexes can silence target mRNAs via imperfect complementarity with sequences located in the 5'-UTR [15–17], coding sequences [18, 19], and most commonly the 3'-UTR [2, 20, 21].
The RNAi pathway is pleotropically functional in many diverse biological processes; and its dysregulation leads to a plethora of pathologies including cancers, metabolic disorders, and infectious diseases [22–24]. In plants, RNAi as a host defense against viral infections has been well-established [25–27]. In vetebrates, the efficacy of RNAi based antiviral defense is debated [28–32], although several findings support the importance of this mechanism [33–40]. Additionally, there is substantial evidence that RNAi is employed by cells as a mean to keep mammalian endogenous retroviruses (i.e. retrotransposons) under check [41–46].
Several studies have reported that cellular miRNAs can modulate HIV-1 replication in human cells. For example, it was found that miR-28, miR-125b, miR-150, miR-223 and miR-382 function to induce HIV-1 latency in T-cells , and that these same miRNAs conferred resistance to HIV-1 infection in blood monocytes . Furthermore, miR-29 has been shown to silence HIV-1 mRNAs that contain Nef sequences [49–51]. Finally, there is accumulating evidence that some cellular miRNAs may indirectly affect HIV-1 through the regulation of cellular proteins, such as Cyclin T1 and PCAF, which are employed for viral replication [52, 53]. These findings are underscored by studies demonstrating, in models of spreading infection, that the over-expression of proteins involved in RNAi decreases viral replication while the knock-down of these proteins increases viral replication [52, 54–56]
Relevant to their interaction with host cells is the question whether retroviruses can encode viral miRNAs. Some have suggested that the potential vulnerability of RNA-genomes to processing by RNAse III enzymes such as Drosha and Dicer might preclude RNA-viruses from containing cis-embedded miRNAs [37, 57–59]. However, it has been shown that the infecting retroviral genome is apparently shielded by RNA-binding proteins rendering it inaccessible to targeting by RNAi factors . Thus, it remains an open question whether a replication competent retroviral genome can encode a viral miRNA. Relevant to this issue, multiple laboratories have reported the processing of the viral TAR RNA into a miRNA-like non-coding RNA in HIV-1 infected cells [61–65]. The complexity of this multitude of findings cautions that a full understanding of the functions of HIV-1 associated non-coding RNAs awaits further investigation .
The current study was undertaken to answer more clearly whether an HIV-1 genome encoding an intragenomic miRNA is precluded from replication competence. We approached this question by creating several HIV-1 molecular genomes that contain discrete cellular miRNAs positioned in the Nef gene. We asked whether the intragenomic presence of the inserted miRNA in HIV-1 prevents viral replication in human cells. Our results showed no absolute preclusion in human cells against the replication of an HIV-1 genome expressing an intragenomic miRNA.
Construction of five discrete HIV-1 molecular clones containing intragenomic miRNA
Expression of the inserted miRNA from the chimeric NL4-3 miR molecular clones
Intragenomic expression of miR28, miR211 and miR326 reduced single cycle HIV-1 infectivity
Reduced infectivity of NL4-3 miR211 and NL4-3 miR326 arises from different mechanisms
The NL4-3 miR21, the NL4-3 miR326 and the NL4-3 miR28 viruses displayed reduced infectivity. We noted that the NL4-3 miR211 virus expressed its corresponding miRNA at a much higher level (>11,000 copies of miR211 per cell) than the NL4-3 miR326 virus (Figure 3). It is possible that an overly efficient processing of a cis-embedded miRNA within a viral RNA genome may deleteriously affect viral replication. Potentially, the reduced replication of the NL4-3 miR211 virus may be explained by this mechanism. By contrast the reduced replication of the NL4-3 miR326 virus may be because the miR326 expressed from Nef led to the silencing of its complementary NL4-3 target sequence (Figure 1A).
We performed a similar analysis using the NL4-3 miR211 virus. In these experiments, 293T cells were transfected with pNL4-3 let7 scr with or without 100 pM of synthetic miR211, and pNL4-3 miR211 was separately transfected into 293T cells with or without 100 pM anti-miR211 antagomir. Next, cell culture supernatants were harvested and measured for infectivity using TZMbl cells (Figure 5B). Interestingly, the production of the NL4-3 let7 scr virus was not silenced by co-transfected synthetic miR211; nor was the infectivity of NL4-3 miR211 virus increased by co-transfected synthetic anti-miR211 antagomirs. These results suggest that the observed reduction in replication of the NL4-3 miR211 virus is unlikely due to miR211-mediated silencing of a putative complementary HIV-1 RNA target sequence (Figure 1A). Further experiments are needed to determine whether the predicted miR211 complementary viral sequence (Figure 1A) is not a competent target or if synthetic miR211 is not efficiently employed as a guide RNA by RISC.
Chimeric NL4-3 miR viruses produce a spreading viral infection in cultured cells
The ability of mammalian DNA viruses to encode viral miRNAs is well accepted. By contrast, it remains debated whether RNA viruses or retroviruses can encode and express viral miRNAs and remain replication competent. The current study tested the hypothesis that an HIV-1 genome with a cis-embedded miRNA can express the miRNA and propagate a spreading infection in cultured T cells.
To check this hypothesis, we constructed several chimeric HIV-1-miRNA molecular genomes with discrete cellular miRNAs cassetted into the viral Nef gene (Figure 2). For most of these chimeric genomes (NL4-3 miR28, NL4-3 miR29b, NL4-3 miR138, NL4-3 miR326 and NL4-3 miR329), the expression of the Nef-inserted miRNAs was several hundred copies per cell (Figure 3). One of the chimeric HIV-1 miRNA virus, NL4-3 miR211, had an unusually high level (>11,000 copies) of miRNA expression (Figure 3). When the viruses were tested for viral infectivity, three (NL4-3 miR28, NL4-3 miR211, and NL4-3 miR326) showed significantly reduced infectivity when compared to the NL4-3 let 7 scr and NL4-3 let7a control viruses (Figure 4).
The NL4-3 miR211 and NL4-3 miR326 viruses were studied in greater detail to understand the reason(s) for reduced infectivity. We explored two possible explanations. One possibility was that the expressed intragenomic miR211 or miR326 miRNAs recognize a cis-HIV-1 target sequence (Figure 1A) and that this miRNA-viral RNA interaction resulted in silencing, reducing viral infectivity. This explanation could be confirmed if an antagomir targeted against either miR211 or the miR326 would rescue the infectivity of the respective NL4-3 miR211 or pNL4-3 miR326 virus. That the infectivity of NL4-3 miR326, but not NL4-3 miR211, was rescued by a sequence-specific antagomir (Figure 5A) supported the interpretation that miRNA-viral RNA silencing explains the reduced infectivity of the former, but not the latter, virus.
What might explain the reduced infectivity of the NL4-3 miR211 virus? A second possibility is that the decreased infectivity may be due to unusually high efficiency of processing miR211 from NL4-3 transcripts that contain cis-inserted miR211 sequence. It may be that some miRNAs are simply better than other miRNAs as substrates for Drosha, Dicer, or both. If all viral RNAs with embedded-miR211 sequence were cleaved by Drosha or Dicer or both, then such events would severely hamper viral protein expression and could explain the severely attenuated infectivity of the NL4-3 miR211 virus. Indeed, we noted that the knockout of Drosha, but not Dicer, rescued NL4-3 miR211 infectivity (Figure 6A) while either knock down improved the infectivity of the NL4-3 miR326 virus (Figure 6A). Taken together with the findings in Figure 5, the results support that two different mechanisms are operative in reducing NL4-3 miR326 and NL4-3 miR211 viral replication (Figure 8).
Our NL4-3 miR211 virus results agree with a similar observation made by Liu et al. in their study of miRNA expression using a single round lentiviral gene delivery vector . Liu et al. also found that the processing by Drosha of some miRNA-cassettes in lentivectors was one of several mechanisms that reduced particle titers. Because Drosha processing is a nuclear event, the likely scenario for reduced NL4-3 miR211 infectivity is the overly robust cleavage of miR211-embedded HIV-1 RNAs transcribed from the integrated proviral DNA genome (Figure 8), not from the cytoplasmic cleavage of miR211-embedded HIV-1 RNA genome. This interpretation agrees with the earlier observation made by Berkhout and colleagues that the infecting lentiviral RNA genome is well-protected from RNAi-mediated silencing .
Finally, we observed that the NL4-3 let7a and the NL4-3 miR326 viruses are capable of a spreading infection in cultured Jurkat T-cells. These results are consistent with no absolute preclusion against a replicating retrovirus encoding and expressing an intragenomic miRNA. Previously, it has been suggested that the proclivity of DNA viruses to replicate in the nucleus and RNA viruses to replicate mostly in the cytoplasm might explain why the former and latter have varying capacity for encoding viral miRNAs. Since a large part of the retroviral life cycle takes place in the nucleus and the genomic retroviral RNA in the cytoplasm is shielded by RNA-binding proteins , these processes may explain why some retroviruses like HIV-1 do produce modest levels of processed non-coding viral RNAs [61, 63, 64]. Other retroviruses like BLV are suggested to potentially encode more non-coding viral RNAs . A recent study reports the expression of a miRNA-like small RNA from the highly structured 3' UTR of West Nile Virus and found that this RNA is supportive of viral replication . If true, this report would represent another example of a miRNA or miRNA-like RNA encoded by an RNA virus. These reports, together with our currently demonstrated replication competence of HIV-1 genomes expressing inserted cellular miRNAs, encourage additional investigation into the nuanced miRNA-encoding capabilities of DNA viruses, RNA viruses, and retroviruses.
293T and TZMbl cells were maintained in DMEM supplemented with L-glutamine, Penicillin/Streptomycin and 10% fetal bovine serum. For transfections, cells were split 24 hours prior to transfection into 6-well plates at 500,000 cells/well. Cells were transfected with lipofectamine LTX (Invitrogen) according to manufacturer's instructions. For production of viral stocks, the supernatant was harvested at 48 hours after transfection. For siRNA transfections, the cells were first transfected with siRNAs and then re-transfected 24 hours later with proviral plasmids. Jurkat T-cell line was maintained in RPMI supplemented with L-glutamine, Penicillin/Streptomycin and 10% fetal bovine serum.
RNA isolation, qRT-PCR and miRNA measurement
RNA was extracted using Trizol reagent (Invitrogen) following manufacturer's protocol. For the determination of mRNA levels, 1 microgram of RNA was used to create cDNA using the SuperScript III First-Strand Synthesis kit (Invitrogen). Following reverse transcription, the samples were diluted 1:50, and 2.5 microliters were used for quantitative PCR in a BioRad CFX96 or CFX384 qPCR machine. All mRNA analyses were normalized to GAPDH. Nucleic acid amplification was tracked by SYBR Green method. For miRNA quantitation, 1 microgram of RNA was processed using QuantiMir (Systems Bioscience Inc.); the resulting tagged cDNA was quantified using miRNA specific primers via qPCR. All miRNA analyses were normalized to the cellular miRNA miR16.
Infections, RT and TZMbl assay
For infection of Jurkat cells, 6 × 106 cells were seeded in 2 ml of media and exposed to the indicated dose of virus supernatant for 24 hours. Cells were then washed and seeded in 10 ml of fresh RPMI, and sampled over time. Replication was measured through use of the RT activity assay: 5 μl of supernatant were added to 50 μl of RT reaction cocktail (60 mM TrisHCl, 75 mM KCl, 5 mM MgCl2, 1.04 mM EDTA, 0.1 NP-40, 5 μg/ml polyA and 0.16 μg/ml oligo dT) and incubated for two hours at 37°C. The reaction mix was spotted on DEAE membrane, washed with SSC, and dried before counting. For TZMbl assay, cells were seeded in a 96 well plate at 15,000 cells/well for 24 hours. Medium was then replaced with fresh RPMI containing serial dilutions of viral supernatant. Twenty-four hours post infection, the cells were washed, fixed and assayed for the presence of β-galactosidase by X-gal enzymatic assay. Blue cells were counted, and the number of infectious units per volume was computed based on the dilution of infecting supernatant.
Cellular DNA from Jurkat T-cells at 8 days post infection was extracted using Qiagen DNA easy kit. PCR for insertion sites was performed, and the resulting fragments were gel purified and cloned into Invitrogen's TopoTA cloning vector before being directly sequenced.
For cloning for miRNA into NL4-3, we followed a previously described procedure . In brief, the sequence of each pre-miRNA was determined by consulting the miRBase for the human miRNA [74, 75]. PCR primers were designed to amplify these sequences with the addition of Sal I (5' end) and Xho I (3' end) to each pre-miRNA. PCR products were cloned into TopoTA vector (Invitrogen) and excised with Sal I and Xho I. Pre-miRNA fragments were then inserted into the Xho I site of a ΔNef shuttle vector, screened for orientation and then moved into the full length pNL4-3 proviral vector to produce the NL4-3 miR clones. All clones were sequenced to verify the proper insertion of the pre-miRNA sequence.
Primers and oligonucleotides for cloning
Primers for generation of Sal I/Xho I miRNA precursors - let7a ATCGTCGACTGGGATGAGGTAGTAGGTTGTATAG/ACTCGAGTAGGAAAGACAGTAGATTGTATAG, miR28 ATCGTCGACGGTCCTTGCCCTCAAGGAGCTCACA/ACTCGAGAGTGCCTGCCCTCCAGGAGCTCACA, miR29b ATCGTCGACCUUCAGGAAGCUGGUUUCAUAUGGU/ACTCGAGCCCCCAAGAACACTGATTTCAAATG, miR138 ATCGTCGACCCCTGGCATGGTGTGGTGGGGCAGC/ACTCGAGCCTGTAGTGTGGTGTGGCCCTGGTG, miR211 ATCGTCGACUCACCUGGCCAUGUGACUUGUGGGC/ACTCGAGCTCCGTGCTGTGGGAAGTGACAACT, miR326 ATCGTCGACCTCATCTGTCTGTTGGGCTG/ACTCGAGTGAATCCGCCTCGGGGCTGG, miR329 ATCGTCGACGGTACCTGAAGAGAGGTTTTCTGGG/ACTCGAGGATACTGGAAAAGAGGTTAACCAGG
Oligonucleotides for the generation of let7 scr through annealing - ATCGTCGACGTTGTTTAGTATAGTTCTATTGCCCCAACTACGGCTAATAAGGTATCGTCC
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