- Open Access
Trans-inhibition of HIV-1 by a long hairpin RNA expressed within the viral genome
© Konstantinova et al; licensee BioMed Central Ltd. 2007
- Received: 06 December 2006
- Accepted: 01 March 2007
- Published: 01 March 2007
Human immunodeficiency virus type 1 (HIV-1) can be inhibited by means of RNA silencing or interference (RNAi) using synthetic short interfering RNAs (siRNAs) or gene constructs encoding short hairpin RNAs (shRNAs) or long hairpin RNAs (lhRNAs). The use of siRNA and shRNA as antiviral therapeutic is limited because of the emergence of viral escape mutants. This problem is theoretically prevented by intracellular expression of lhRNAs generating multiple siRNAs that target the virus simultaneously, thus reducing the chance of viral escape. However, gene constructs encoding lhRNA molecules face problems with delivery to the right cells in an infected individual. In order to solve this problem, we constructed an HIV-1 variant with a 300 bp long hairpin structure in the 3' part of the genome corresponding to the Nef gene (HIV-lhNef).
Intriguingly, HIV-lhNef potently inhibited wild-type HIV-1 production in trans. However, HIV-lhNef demonstrated a severe production and replication defect, which we were able to solve by selecting spontaneous virus variants with truncated hairpin structures. Although these escape variants lost the ability to trans-inhibit HIV-1, they effectively outgrew the wild-type virus in competition experiments in SupT1 cells.
Expression of the lhNef hairpin within the HIV-1 genome results in potent trans-inhibition of wild-type HIV-1. Although the mechanism of trans-inhibition is currently unknown, it remains of interest to study the molecular details because the observed effect is extremely potent. This may have implications for the development of virus strains to be used as live-attenuated virus vaccines.
- Hairpin Structure
- Escape Variant
- SupT1 Cell
- Photinus Luciferase
RNA interference (RNAi) has been used to inhibit the replication of a wide range of viruses including the human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV), dengue virus, poliovirus, influenza virus A, coronaviruses, herpesviruses, and picornaviruses [1, 2]. Due to its sequence specificity, RNAi is a potentially selective method for intracellular immunization against HIV-1 infection. RNAi-mediated suppression of HIV-1 replication has been accomplished by synthetic small interfering RNAs (siRNAs) in a transient manner [3–6] and by shRNA expression vectors in stably transfected cells [7–9]. Despite potent inhibition, the use of both approaches as therapeutic antiviral is limited because of the rapid emergence of HIV-1 escape mutants [9–11]. Strategies to reduce the chance of viral escape include the simultaneous use of multiple siRNAs [12, 13], the intracellular expression of a second generation of escape-anticipating shRNAs , or microRNA-based double-stranded RNAs (miRNAs), which do not require perfect sequence complementarity for inhibition [15, 16].
An alternative method to inhibit HIV-1 is the use of gene constructs encoding HIV-1-specific long hairpin RNAs (lhRNAs, transcripts folding an extended hairpin structure) or long double-stranded RNAs (dsRNAs, two complementary transcripts that form an extended duplex). These molecules should yield multiple effective siRNAs upon intracellular processing [6, 17, 18]. However, lhRNA approaches raise concerns about induction of the dsRNA-triggered interferon (IFN) response. Others and we have shown that endogenously expressed lhRNA and dsRNA can inhibit HIV-1 production without induction of the innate antiviral response [18–21]. In fact, most reports on IFN induction by long dsRNAs in mammalian cells are based on transfection of cells with in vitro synthesized dsRNAs [22, 23]. Apparently, endogenously produced dsRNA is less active than exogenous dsRNA in inducing the IFN response.
Several antiviral approaches using extended lhRNA and long dsRNA molecules have been reported in plant and insect cells that lack the innate antiviral IFN response. Transient expression of DNA constructs encoding virus-specific dsRNA in plant protoplasts or insect cells partially protects the cells from infection by the homologous virus [23, 24]. Stable expression of such constructs renders the cells resistant to infection [25, 26]. lhRNA can inhibit HIV-1 production under certain conditions, without induction of the IFN response [6, 17, 18]. Ideally, a single lhRNA should generate multiple effective siRNAs upon intracellular processing, providing more durable inhibition of HIV-1 than a single shRNA. An additional advantage of lhRNA inhibitors is that it does not require pre-determination of the optimal shRNAs and corresponding HIV-1 target sequences because multiple effective siRNAs will be produced. A potential disadvantage of the use of lhRNA as therapeutic is that the generation of multiple siRNAs will be more likely to cause off-target effects.
We have previously reported strong inhibition of HIV-1 production using gene constructs encoding HIV-specific lhRNAs and dsRNA in transient transfection assays . An alternative for stable expression of shRNAs is a conditionally replicating HIV-1-based virus, which was previously used by us to deliver an antiviral shRNA cassette into HIV-1 susceptible target cells . In the current study, we constructed the HIV-lhNef variant, which contains a 300 bp extended hairpin structure at the 3' genome position of the Nef gene of the otherwise wild-type HIV-1. We tested this HIV-lhNef for its capacity to inhibit the production of wild-type virus. Intriguingly, HIV-lhNef potently inhibited wild-type HIV-1 production in trans. However, HIV-lhNef demonstrated a severe production and replication defect, which we were able to solve by selecting spontaneous, escape viruses.
The HIV-lhNef variants
Trans-inhibition of wild-type HIV-1
Replication properties of the escape viruses
Virus competition between HIV-1 and the HIV-lhNef escape variants
As a more sensitive assay for possible trans-inhibition, we tested the individual AS variants in a direct competition with wild-type HIV-1 in PBMC. Cells were infected with an equimolar mixture of the two viruses (based on CA-p24) that were produced in HEK293T cells, and virus was passaged to fresh cells at the peak of infection. Virus replication was monitored by CA-p24 ELISA and visual inspection for syncytia. Cellular DNA was extracted at several times post-infection and the proviral Nef region was PCR amplified with primers tTA1 and CN1 (see Fig. 1). Since the PCR products will differ in size for wild-type HIV-1 and each AS variant, both competitors can be detected in the same sample by subsequent agarose gel electrophoresis of the DNA fragments. The outgrowth of a particular virus was verified by cloning and sequencing of the PCR products from the last time-point sample (results not shown).
Pairwise virus competition experiments
Virus mix a
Mechanism of trans-inhibition by HIV-lhNef and the AS variants
In this study, we show that HIV-1 expressing an excessively stable 300 bp lhNef hairpin potently inhibits wild-type HIV-1 in trans. However, HIV-lhNef did not replicate to detectable levels in HIV-1 target cells, probably because steps of its replication cycle are affected by the hairpin insertion, e.g. RNA splicing, RNA nuclear export or mRNA translation. Moreover, HIV-lhNef may cause degradation of its own mRNA due to processing by the RNAi machinery. We postulate that the lhNef hairpin may induce an antiviral response against wild-type HIV-1 either through a sequence-specific RNAi mechanism or through aberrant HIV-1 transcripts lacking the 3' non-coding region and polyA tail that induce a potent HIV-1-specific RNA silencing response in cells. Tests with reporter constructs do not support a sequence-specific RNAi effect, although the effect induced by lhNef is HIV-1 specific. The mechanism of trans-inhibition is currently unknown, but it remains of interest to study the molecular details because the observed inhibition is extremely potent. We are currently dissecting the mechanism of inhibition by testing HIV-lhNef variants that lack important RNA signals (TAR, polyA, PAS, PBS, DIS, SD, psi).
Although HIV-lhNef is a potent inhibitor of wild-type HIV-1, its inability to replicate precluded long-term inhibition experiments. After prolonged culturing of HIV-lhNef, replicating variants emerged through recombination events that introduce large truncations in the lhNef hairpin structure and therefore deletions in the Nef gene. Interestingly, all AS variants inherit a part of the hairpin structure in their genome, there were no perfect deletions of the entire lhNef region. One could speculate that the remaining secondary RNA structures are actively selected because they render the viral genome less susceptible for degradation by the RNAi machinery .
Nef represents a pathogenicity factor that disorders adaptive immunity by down regulating CD4 and MHC-1 receptors, by inhibiting T-cell chemotaxis, and by inducing apoptosis in bystander T-cells, and hence plays a major role in the destruction of the host immune system [32, 33]. It has been suggested that any therapeutic intervention aimed at either completely blocking or at least partially reducing the expression of Nef during HIV-1 infection would likely enhance the ability of the immune system to fight HIV infection . Humans infected with Nef-defective HIV-1 strains show low viral loads and no or very slow disease progression and represent long-term non-progressors or long-term slow-progressors [31, 35]. Moreover, macaques vaccinated with a SIV strain that only lacks Nef are better protected against superinfection than macaques vaccinated with a SIV strain lacking the three accessory genes Nef, Vpr and Vpx . The cross-protection conferred by the attenuated SIV strains appears not to be based on stimulation of the adaptive immune system, but on other (unknown) mechanisms . In this study, we demonstrate effective outgrowth of the Nef-minus AS escape variants in competitions with wild-type HIV-1. This result was obtained in the SupT1 T cell line, which does not provide a clear Nef-phenotype. We think that this outcompetition is related to the presence of the genomic hairpin structure, because no such effect was observed for the control Nef-deletion virus R1. These in vitro results may relate to the superior protection with Nef-deleted viruses in vivo.
Successful expression of a shRNA from a replicating viral vector has been shown for Rous sarcoma virus (RSV) in avian cells . We previously described inhibition of HIV-1 replication by a conditional-live HIV-rtTA virus that expresses a shRNA against the Nef gene . This approach is especially suitable for targeting cells that are susceptible to HIV-1 infection. However, HIV-1 escape variants will emerge rapidly under shNef pressure . Expression of multiple shRNAs or a single lhRNA from such a vector could prevent viral escape because multiple precise mutations should occur. Use of a murine leukemia virus (MLV) can also be used for efficient and stable delivery of anti-HIV-1 shRNA . However, MLV can replicate only in actively dividing cells, which limits its application as a therapeutic virus.
One ideal property of replicating HIV-1-based viral vectors is that they specifically target HIV-1 susceptible cells. The replication-competent AS escape variants lost the trans-inhibitory properties of HIV-lhNef, but effectively outcompeted wild-type HIV-1 in T cells. It is important to asses what is the minimum length of a hairpin that can mediate trans-inhibition of wild type HIV-1. We have previously shown that a 19 nt shRNA expressed from conditionally replicating HIV-1-based virus, can inhibit viral replication . Any hairpin longer than that should in theory mediate trans-inhibition of HIV-1. Further research is needed to see if we can design constructs that are replication competent, yet remain a potent trans-inhibitor of HIV-1. For example, a lhRNA variant with G-U wobbles could be designed, which will destabilize the RNA structure and therefore stimulate viral replication. Such an approach using 90–100 bp lhRNA molecules has been suggested as a means for intracellular immunisation against HBV and HCV, without evoking non-specific IFN responses [19, 40]. Such constructs could form the basis for an attenuated virus vaccine and anti-HIV therapeutic virus in one.
DNA constructs and proviral DNA analysis
The full-length molecular HIV-1 clone LAI  (Accession number AF33819.3) was used to produce wild-type virus. HIV-lhNef has a 93 nt deletion in the 5' region of Nef and a 300 bp inverted repeat within the Nef gene (Fig. 1). The construction of the HIV-lhNef mutant and the generation of molecular clones of HIV-lhNef escape virus variants AS44, AS19, AS15, AS11 and AS8 has been described previously . The HIV-1 mutant R1 has a 106 nt deletion in Nef (+8513 to +8619) and has been described previously . HIV-CMV and HIV-asCMV were constructed by inserting the CMV promoter in sense or antisense orientation at position +8069 of the LAI genome. pSuper-shGag and pSuper-shNef with the H1 polymerase III promoter have been described previously . Nucleotide numbers refer to the position on the genomic HIV-1 RNA transcript, with +1 being the capped G residue. Plasmid pGL3-Nef (Luc-Nef), in which 250 nt from the Nef gene was placed downstream of the Photinus luciferase reporter gene has been described previously .
For cellular DNA isolation, cells were pelleted for 4 min at 4000 rpm and solubilized in 150 μl lysis buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.5 % Tween 20) and 200 μg/ml proteinase K (Roche) at 56°C for 1 h and at 95°C for 10 min. Proviral DNA sequences were PCR amplified from 5 μl cellular lysate using the 5' Env primer tTA1-AD (+8269 to +8289, ACA GCC ATA GCA GTA GCT GAG) and 3' U5 primer CN1 (+9283 to +9253, GGT CTG AGG GAT CTC TAG TTA CCA GAG TC) (Fig. 1). PCR amplification was performed in a 50 μl reaction containing 1 × PCR amplification buffer (Invitrogen), 0.5 mM MgCl2, 25 pmol of each primer, 0.2 mM dNTPs and 2.5 units of AmpliTaq DNA polymerase (Perkin Elmer Applied Biosystem). The PCR program was as follows: 95°C for 5 min, 30 cycles of 30 sec at 94°C, 30 sec at 55°C, 30 sec at 72°C, and a final extension for 7 min at 72°C. The PCR products were separated on a 1% agarose gel stained with ethidium bromide and compared to a standard DNA size marker (Eurogentec). HIV-lhNef could not be amplified with this PCR due to the extended inverted repeat that may form a cruciform DNA structure. The RNA structures formed by HIV-lhNef and the AS escape variants were predicted with the Mfold program .
Cells, DNA transfection and virus infection
Human embryonic kidney (HEK) 293T cells were grown as a monolayer in DMEM (Invitrogen) supplemented with 10% FCS, minimal essential medium nonessential amino acids, penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C and 5% CO2. One day before transfection, cells were trypsinized, resuspended in DMEM and seeded in 24-well plates at a density of 1.5 × 105 cells per well. Cells were co-transfected with 100 – 500 ng DNA with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. 1 ng pRL plasmid (Promega), expressing Renilla luciferase (RL) from the CMV promoter, was added as an internal control for cell viability and transfection efficiency. In all transfection experiments we have controlled for the DNA input by adding pBluescript (Promega) to reach identical DNA concentrations.
Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by standard Ficoll-Hypaque density centrifugation, activated with phytohemagglutinin (3 μg/ml), and cultured in complete RPMI 1640 medium (Invitrogen) with IL-2 (100 U/ml). Cells (5 × 106) were transfected with 10 μg of the proviral DNA by electroporation in 250 μl RPMI with 20% FCS in 0.4-cm cuvettes at 250 V and 960 μF, and 1 × 106 fresh cells and 5 ml complete RPMI with IL-2 were added afterwards. PBMCs were infected with a fixed amount (5 ng CA-p24) of 293T-produced virus and maintained for up to 4 weeks. Half of the culture medium was replaced every 4 days by fresh complete RPMI 1640 medium with IL-2 and freshly activated PBMC (2 × 106) were added at every 2nd addition.
The human T cell line SupT1 was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) (Hybond), penicillin (100 U/ml) and streptomycin (100 μg/ml) at 37°C and 5% CO2. Cells were cultured in 25 cm2 flasks and split 1 to 10 twice a week. Cells were infected with equal amounts (5 ng CA-p24) of 293T-produced virus. When HIV-induced cytopathic effects were observed, virus replication was maintained by passage of the cell-free culture supernatant onto uninfected SupT1 cells. At each passage, cell and supernatant samples were stored at -70°C. Virus spread was followed by CA-p24 ELISA on the culture supernatant as described previously  and Renilla expression was measured with the Renilla luciferase assay system (Promega).
HIV-1 RNA research in the Berkhout lab is sponsored by ZonMw (VICI grant), NWO-CW (TOP grant) and Senter-NOVEM (TS grant with Viruvation). The authors thank Stephan Heynen for performing CA-p24 Elisa and Nienke Westerink for providing the PBMC.
- Haasnoot PCJ, Cupac D, Berkhout B: Inhibition of virus replication by RNA interference. J Biomed Sc. 2003, 10: 607-616.View ArticleGoogle Scholar
- Berkhout B, Haasnoot J: The interplay between virus infection and the cellular RNA interference machinery. FEBS Lett. 2006, 580: 2896-2902. 10.1016/j.febslet.2006.02.070.View ArticlePubMedGoogle Scholar
- Capodici J, Kariko K, Weissman D: Inhibition of HIV-1 infection by small interfering RNA-mediated RNA interference. J Immunol. 2002, 169: 5196-5201.View ArticlePubMedGoogle Scholar
- Jacque JM, Triques K, Stevenson M: Modulation of HIV-1 replication by RNA interference. Nature. 2002, 418: 435-438. 10.1038/nature00896.View ArticlePubMedGoogle Scholar
- Novina CD, Murray MF, Dykxhoorn DM, Beresford PJ, Riess J, Lee SK, Collman RG, Lieberman J, Shankar P, Sharp PA: siRNA-directed inhibition of HIV-1 infection. Nat Med. 2002, 8: 681-686.PubMedGoogle Scholar
- Park WS, Miyano-Kurosaki N, Hayafune M, Nakajima E, Matsuzaki T, Shimada F, Takaku H: Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference. Nucleic Acids Res. 2002, 30: 4830-4835. 10.1093/nar/gkf627.PubMed CentralView ArticlePubMedGoogle Scholar
- Anderson J, Banerjea A, Akkina R: Bispecific short hairpin siRNA constructs targeted to CD4, CXCR4, and CCR5 confer HIV-1 resistance. Oligonucleotides. 2003, 13: 303-312. 10.1089/154545703322616989.View ArticlePubMedGoogle Scholar
- Boden D, Pusch O, Silbermann R, Lee F, Tucker L, Ramratnam B: Enhanced gene silencing of HIV-1 specific siRNA using microRNA designed hairpins. Nucleic Acids Res. 2004, 32: 1154-1158. 10.1093/nar/gkh278.PubMed CentralView ArticlePubMedGoogle Scholar
- Das AT, Brummelkamp TR, Westerhout EM, Vink M, Madiredjo M, Bernards R, Berkhout B: Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition. J Virol. 2004, 78: 2601-2605. 10.1128/JVI.78.5.2601-2605.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Boden D, Pusch O, Lee F, Tucker L, Ramratnam B: Human immunodeficiency virus type 1 escape from RNA interference. J Virol. 2003, 77: 11531-11535. 10.1128/JVI.77.21.11531-11535.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Westerhout EM, Ooms M, Vink M, Das AT, Berkhout B: HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome. Nucleic Acids Res. 2005, 33: 796-804. 10.1093/nar/gki220.PubMed CentralView ArticlePubMedGoogle Scholar
- Berkhout B: RNA interference as an antiviral approach: targeting HIV-1. Curr Opin Mol Ther. 2004, 6: 141-145.PubMedGoogle Scholar
- Ter Brake O, Konstantinova P, Ceylan M, Berkhout B: Silencing of HIV-1 with RNA Interference: a multiple shRNA approach. Mol Ther. 2006, 14: 883-892. 10.1016/j.ymthe.2006.07.007.View ArticlePubMedGoogle Scholar
- Ter Brake O, Berkhout B: A novel approach for inhibition of HIV-1 by RNA interference: counteracting viral escape with a second generation of siRNAs. Journal of RNAi and Gene Silencing. 2005, 1(2): 56-65.Google Scholar
- Yeung ML, Bennasser Y, Myers TG, Jiang G, Benkirane M, Jeang KT: Changes in microRNA expression profiles in HIV-1-transfected human cells. Retrovirology. 2005, 2: 81-10.1186/1742-4690-2-81.PubMed CentralView ArticlePubMedGoogle Scholar
- Hariharan M, Scaria V, Pillai B, Brahmachari SK: Targets for human encoded microRNAs in HIV genes. Biochem Biophys Res Commun. 2005, 337: 1214-1218. 10.1016/j.bbrc.2005.09.183.View ArticlePubMedGoogle Scholar
- Yamamoto T, Omoto S, Mizuguchi M, Mizukami H, Okuyama H, Okada N, Saksena NK, Brisibe EA, Otake K, Fuji YR: Double-stranded nef RNA interferes with human immunodeficiency virus type 1 replication. Microbiol Immunol. 2002, 46: 809-817.View ArticlePubMedGoogle Scholar
- Konstantinova P, de Vries W, Haasnoot J, Ter Brake O, de Haan P, Berkhout B: Inhibition of human immunodeficiency virus type 1 by RNA interference using long-hairpin RNA. Gene Ther. 2006, 13: 1403-1413. 10.1038/sj.gt.3302786.View ArticlePubMedGoogle Scholar
- Akashi H, Miyagishi M, Yokota T, Watanabe H, Hino T, Nishina K, Kohara M, Taira K: Escape from the interferon response associated with RNA interference using vectors that encode long modified hairpin-RNA. Molecular BioSystems. 2005, 1: 382-390. 10.1039/b510159j.View ArticlePubMedGoogle Scholar
- Matsumoto S, Miyagishi M, Akashi H, Nagai R, Taira K: Analysis of double-stranded RNA-induced apoptosis pathways using interferon-response noninducible small interfering RNA expression vector library. J Biol Chem. 2005, 280: 25687-25696. 10.1074/jbc.M412784200.View ArticlePubMedGoogle Scholar
- Nishitsuji H, Kohara M, Kannagi M, Masuda T: Effective suppression of human immunodeficiency virus type 1 through a combination of short- or long-hairpin RNAs targeting essential sequences for retroviral integration. J Virol. 2006, 80: 7658-7666. 10.1128/JVI.00078-06.PubMed CentralView ArticlePubMedGoogle Scholar
- Elbashir SM, Lendeckel W, Tuschl T: RNA interference is mediated by 21- and 22-nucleotide RNAs. Genes Dev. 2001, 15: 188-200. 10.1101/gad.862301.PubMed CentralView ArticlePubMedGoogle Scholar
- Caplen NJ, Zheng Z, Falgout B, Morgan RA: Inhibition of viral gene expression and replication in mosquito cells by dsRNA-triggered RNA interference. Mol Ther. 2002, 6: 243-251. 10.1006/mthe.2002.0652.View ArticlePubMedGoogle Scholar
- Qi Y, Zhong X, Itaya A, Ding B: Dissecting RNA silencing in protoplasts uncovers novel effects of viral suppressors on the silencing pathway at the cellular level. Nucleic Acids Res. 2004, 32: e179-10.1093/nar/gnh180.PubMed CentralView ArticlePubMedGoogle Scholar
- Waterhouse PM, Graham MW, Wang MB: Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA. 1998, 95: 13959-13964. 10.1073/pnas.95.23.13959.PubMed CentralView ArticlePubMedGoogle Scholar
- Adelman ZN, Sanchez-Vargas I, Travanty EA, Carlson JO, Beaty BJ, Blair CD, Olson KE: RNA silencing of dengue virus type 2 replication in transformed C6/36 mosquito cells transcribing an inverted-repeat RNA derived from the virus genome. J Virol. 2002, 76: 12925-12933. 10.1128/JVI.76.24.12925-12933.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Westerhout EM, Vink M, Haasnoot PC, Das AT, Berkhout B: A conditionally replicating HIV-based vector that stably expresses an antiviral shRNA against HIV-1 replication. Mol Ther. 2006, 14: 268-275. 10.1016/j.ymthe.2006.03.018.View ArticlePubMedGoogle Scholar
- Konstantinova P, de Haan P, Das AT, Berkhout B: Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1. Nucleic Acids Res. 2006, 34: 2206-2218. 10.1093/nar/gkl226.PubMed CentralView ArticlePubMedGoogle Scholar
- Beerens N, Groot F, Berkhout B: Stabilization of the U5-leader stem in the HIV-1 RNA genome affects initiation and elongation of reverse transcription. Nucleic Acids Res. 2000, 28: 4130-4137. 10.1093/nar/28.21.4130.PubMed CentralView ArticlePubMedGoogle Scholar
- de Ronde A, Klaver B, Keulen W, Smit L, Goudsmit J: Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes. Virol. 1992, 188: 391-395. 10.1016/0042-6822(92)90772-H.View ArticleGoogle Scholar
- Deacon NJ, Tsykin A, Solomon A, Smith K, Ludford-Menting M, Hooker DJ, McPhee DA, Greenway AL, Ellett A, Chatfield C, Lawson VA, Crowe S, Maerz A, Sonza S, Learmont J, Sullivan JS, Cunningham A, Dwyer D, Dowton D, Mills J: Genomic structure of an attenuated quasi species of HIV-1 from blood transfusion donor and recipients. Science. 1995, 270: 988-991. 10.1126/science.270.5238.988.View ArticlePubMedGoogle Scholar
- Swingler S, Brichacek B, Jacque JM, Ulich C, Zhou J, Stevenson M: HIV-1 Nef intersects the macrophage CD40L signalling pathway to promote resting-cell infection. Nature. 2003, 424: 213-219. 10.1038/nature01749.View ArticlePubMedGoogle Scholar
- Schiavoni I, Trapp S, Santarcangelo AC, Piacentini V, Pugliese K, Baur A, Federico M: HIV-1 Nef enhances both membrane expression and virion incorporation of Env products. A model for the Nef-dependent increase of HIV-1 infectivity. J Biol Chem. 2004, 279: 22996-23006. 10.1074/jbc.M312453200.View ArticlePubMedGoogle Scholar
- Yamamoto T, Miyoshi H, Yamamoto N, Yamamoto N, Inoue J, Tsunetsugu-Yokota Y: Lentivirus vectors expressing short hairpin RNAs against the U3-overlapping region of HIV nef inhibit HIV replication and infectivity in primary macrophages. Blood. 2006, 108: 3305-3312. 10.1182/blood-2006-04-014829.View ArticlePubMedGoogle Scholar
- Rhodes DI, Ashton L, Solomon A, Carr A, Cooper D, Kaldor J, Deacon N: Characterization of three nef-defective human immunodeficiency virus type 1 strains associated with long-term nonprogression. Australian Long-Term Nonprogressor Study Group. J Virol. 2000, 74: 10581-10588. 10.1128/JVI.74.22.10581-10588.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Johnson RP: Live attenuated AIDS vaccines: hazards and hopes. Nature Medicine. 1999, 5: 154-155. 10.1038/5515.View ArticlePubMedGoogle Scholar
- Stebbings R, Berry N, Stott J, Hull R, Walker B, Lines J, Elsley W, Brown S, Wade-Evans A, Davis G, Cowie J, Sethi M, Almond N: Vaccination with live attenuated simian immunodeficiency virus for 21 days protects against superinfection. Virol. 2004, 330: 249-260. 10.1016/j.virol.2004.09.026.View ArticleGoogle Scholar
- Bromberg-White JL, Webb CP, Patacsil VS, Miranti CK, Williams BO, Holmen SL: Delivery of short hairpin RNA sequences by using a replication-competent avian retroviral vector. J Virol. 2004, 78: 4914-4916. 10.1128/JVI.78.9.4914-4916.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Sliva K, Schnierle BS: Stable integration of a functional shRNA expression cassette into the murine leukemia virus genome. Virol. 2006, 351: 218-225.View ArticleGoogle Scholar
- Weinberg MS, Ely A, Barichievy S, Crowther C, Mufamadi S, Carmona S, Arbuthnot P: Specific Inhibition of HBV Replication In Vitro and In Vivo With Expressed Long Hairpin RNA. Mol Ther. 2007, 15 (3): 534-41. 10.1038/sj.mt.6300077.View ArticlePubMedGoogle Scholar
- Peden K, Emerman M, Montagnier L: Changes in growth properties on passage in tissue culture of viruses derived from infectious molecular clones of HIV-1LAI, HIV-1MAL, and HIV-1ELI. Virol. 1991, 185: 661-672. 10.1016/0042-6822(91)90537-L.View ArticleGoogle Scholar
- Zuker M: Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 2003, 31: 3406-3415. 10.1093/nar/gkg595.PubMed CentralView ArticlePubMedGoogle Scholar
- Jeeninga RE, Hoogenkamp M, Armand-Ugon M, de Baar M, Verhoef K, Berkhout B: Functional differences between the long terminal repeat transcriptional promoters of HIV-1 subtypes A through G. J Virol. 2000, 74: 3740-3751. 10.1128/JVI.74.8.3740-3751.2000. [http://PM:10729149]PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.